The kidney contains several distinct epithelia that, in their aggregate function, are responsible for formation of the urine. We are studying the roles of these epithelia in the regulation of the excretion of water, urea, ammonium, bicarbonate, sodium, potassium, and chloride. The general approach is to dissect the epithelia from the kidney and to study their functions in vitro. The data are analyzed and integrated using mathematical models of transport in the kidney. Experiments in the cortical collecting duct of rat showed a large fraction of active NaCl absorption occurs via a thiazide-sensitive electroneutral transport pathway which is regulated by atrial natriuretic factor (ANF). Studies are underway, using the polymerase chain reaction in single microdissected rat nephron segments, to localize along the nephron the specific mRNA that codes for the ANF receptor-guanylate cyclase protein. Isolated perfused tubule studies have demonstrated the presence of a vasopressin-regulated urea transporter in the apical and basolateral membranes of the rat inner medullary collecting duct. In preliminary studies, the transporter has been expressed in Xenopus oocytes by injecting them with mRNA from rabbit renal inner medulla (collaboration with M. Hediger, Boston). We have developed and validated a high time-resolution spectrofluorometric method to measure urea transport in perfused tubules. Studies are continuing to map the NH3, NH4+, and bicarbonate permeabilities along the nephron with a long term goal of understanding the mechanism by which acid-base excretion is regulated by the kidney. Mapping of the activities of ammoniagenic enzymes along the nephron have been completed.